DNA topoisomerases are enzymes that affect the topology or structure of DNA. More specifically, these enzymes have the ability to introduce supercoils into DNA molecules or relax the DNA molecules; they can catenate or decatenate circular DNA or they can knot or unknot DNA (Schmid et al., 1993, BioEssays 15 (No. 7): 445-449). The DNA topoisomerases act by catalyzing the breakage and rejoining of the DNA phosphodiester backbone (Wang, 1985, Annu. Rev. Biochem. 54: 665-697; Vosberg, 1985, Curr. Top. Microbiol. Immunol. 114: 19-102). These reactions, together with an intervening strand passage event, allow the topoisomerases to alter DNA topology. In fact, correct topoisomerase function is necessary for such basic cellular processes as DNA replication and transcription.
More specifically, the DNA topoisomerases are classified into two types. Type I topoisomerases act by causing a transient break in one strand of the double-stranded DNA and passing one strand of DNA through another, thereby allowing for the relaxation of supercoiled DNA and decatenation of interlocked circular DNA molecules (Schmid et al., supra). In contrast, the Type II DNA topoisomerases alter DNA topology by causing transient breaks in both strands of a double-stranded DNA, allowing the passage of one double-stranded DNA molecule through another. Like the Type I DNA topoisomerases, the Type II topoisomerases also allow the relaxation and decatenation of DNA; however, one bacterial Type II topoisomerase, DNA gyrase, also has the ability to introduce negative supercoils into relaxed DNA. In either type of topoisomerase, after the strand passage event, the final step of the reaction is the rejoining or ligation of the DNA break(s).
Analysis of the mechanism of action of the DNA topoisomerases on the molecular level indicates that these enzymes introduce breaks in the DNA molecule by forming a covalent phosphotyrosine bond between a specific tyrosine amino acid residue present on the topoisomerase and the phosphodiester backbone of the DNA. Thus, an intermediate in the catalytic reaction of these enzymes is a covalently-linked enzyme-DNA complex, sometimes termed the cleavable complex (see, e.g., Liu, 1990, DNA Topology and Its Biological Effects, Cozzarelli and Wang (eds.), Chapter 14, pp. 371-372, Cold Spring Harbor Press). It is this cleavable complex that forms the molecular target for therapeutic compounds that can interact with this intermediate, stabilizing it, i.e., trapping it, such that the subsequent DNA strand ligation step of the reaction cannot be completed.
In bacteria, two Type I topoisomerase enzymes have been described: Topoisomerase I (Topo I) and Topoisomerase III (Topo III). Topo I, encoded by the topA gene, is responsible for the major DNA relaxing activity of the cell, and catalyzes the relaxation of DNA through the sequential breakage-strand-passage-religation cycle common to all topoisomerase enzymes. Topo III represents a minor activity encoded by the topB gene, is less well-characterized and its role in vivo is less clear, although the molecule appears to possess decatenation activity (Scmid et al., supra at p. 447). Bacteria also have two Type II topoisomerases: DNA gyrase (Topo II) and Topoisomerase IV (Topo IV). As noted supra, DNA gyrase catalyzes negative supercoiling in DNA; Topo IV has been found to catalyze decatenation of interlinked DNA, e.g., subsequent to DNA replication, as well play a role in DNA relaxation. Although Topo IV shares extensive homology with DNA gyrase (40-50% amino acid identity), it differs from DNA gyrase in the activities it can catalyze. For example, Topo IV cannot couple the hydrolysis of ATP to the process of DNA supercoiling as gyrase can, but it is able to relax DNA in an ATP-stimulated fashion.
In contrast to the bacteria, eukaryotes, such as yeast and humans, possess a single Type I DNA topoisomerase (see, e.g., Wang, J. C., 1987, J. Biochem. Biophys. Acta. 909: 1-9). Furthermore, this eukaryotic topoisomerase is distinct from bacterial Topo I in both structure and function. For example, the bacterial and eukaryotic Topo I enzymes share no amino acid sequence homology. In addition, the eukaryotic enzyme shows a preference for binding double-stranded DNA and catalyzes its reaction by forming a covalent 3'-phosphodiester intermediate. In contrast, the bacterial Topo I enzyme shows a preference for binding at the junction of double and single-stranded regions and catalyzes its reaction by forming a 5'-phosphodiester intermediate (see, e.g., Taylor and Menzel, 1995, Gene 167: 69-74). Finally, it is of interest to note that, while the human Topo I enzyme is the target of the antitumor drug camptothecin (CPT), the bacterial enzyme is resistant to that drug. Eukaryotes also possess a Type II DNA topoisomerase that demonstrates some structural and evolutionary relationship to bacterial gyrase (Wang, 1994, Advances in Pharmacology, Volume 29A, Liu (ed.), Academic Press).
While the bacterial Type I topoisomerase, Topo III, appears to be non-essential to the viability of the cell (Hiasa, et al., 1994, J. Biol. Chem. 269: 2093-2099), Topo I is essential in an otherwise normal bacterial cell. Nevertheless, the topA gene may be deleted or otherwise rendered inactive, provided that certain second site compensatory mutations are present in the topA.sup.- strains, leading some to question the essential nature of the bacterial Topo I enzyme (Pruss et al., 1982, Cell 31: 35-42; DiNardo et al., 1982, Cell 31: 43-51).
Second site mutations that compensate for the loss of topA do so by altering the activity or levels of other topoisomerase enzymes in the mutant host cell. For example, mutations that compensate for the loss of bacterial Topo I have been mapped to the two genes (gyrA and gyrB) that encode the bacterial Type II DNA topoisomerase, DNA gyrase. These mutations have been shown to reduce the levels and activity of DNA gyrase, suggesting a balance between the competing activities of topoisomerases that introduce DNA supercoils (i.e., DNA gyrase) and those that remove or relax DNA supercoils (e.g., Topo I).
Still other mutations that compensate for the loss of topA in a topA mutant have been mapped to a region of the chromosome encoding the parC and parE genes which encode the subunits of the bacterial Type II topoisomerase, Topo IV. The majority of these compensatory mutations are duplications of the parC,E region of the chromosome. Such duplications are believed to increase the copy number, and hence the level, of Topo IV within the bacterial cell. It has also been shown that extra copies of Topo IV-encoding sequences on a plasmid are able to compensate for a loss of topA.
As noted supra, compounds that can trap the cleavable complex intermediate formed during the catalytic reaction of the DNA topoisomerases represent important drugs. For example, compounds that trap the covalent intermediates involved in the catalytic reactions of the eukaryotic DNA topoisomerases have been shown to represent important anticancer drugs; e.g., the anticancer drugs Etoposide and Adriamycin trap the covalent intermediate of human Type II topoisomerases. As noted supra, camptothecins, another important class of anticancer drug, trap the DNA intermediate of human Type I topoisomerase but do not affect the bacterial Type I topoisomerases (Drlica, K. et al., Biochem. 27: 2253-2259). In the area of antibacterial drugs, a major class of antibiotics, the quinolones, have been shown to trap the cleavable complex of the bacterial DNA gyrase enzyme. The quinolones have also been shown to trap the intermediate formed during the catalytic reaction of Topo IV (Drlica et al., September 1997, Microbiology and Molecular Biology Reviews, 61 (No. 3): 377-392).
Model systems for the study of eukaryotic Type I DNA topoisomerases have been reported (Nitiss, J. et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7501-7505; M. -A., Bjornsti et al., 1989, Cancer Research 49: 6318-6323; Menzel, R. et al., U.S. Pat. No. 5,656,495). To date, however, no model systems exist for the systematic identification of compounds which target the bacterial DNA topoisomerases. Furthermore, no bacterial assays have been reported which utilize varying levels of bacterial DNA topoisomerase targets within bacterial cells to screen for drugs that interact with those enzymes in the identification of antibacterial compounds, as disclosed herein. The methods and compositions of this invention can lead to the identification and use of important new antibacterial compounds.